Efficient PD-L1 imaging of murine glioblastoma with FUS-aided immunoPET by leveraging FcRn-antibody interaction

Rationale: The passage of antibodies through the blood-brain barrier (BBB) and the blood-tumoral barrier (BTB) is determinant not only to increase the immune checkpoint inhibitors efficacy but also to monitor prognostic and predictive biomarkers such as the programmed death ligand 1 (PD-L1) via immunoPET. Although the involvement of neonatal Fc receptor (FcRn) in antibody distribution has been demonstrated, its function at the BBB remains controversial, while it is unknown at the BTB. In this context, we assessed FcRn's role by pharmacokinetic immunoPET imaging combined with focused ultrasounds (FUS) using unmodified and FcRn low-affinity IgGs targeting PD-L1 in a preclinical orthotopic glioblastoma model. Methods: Transcranial FUS were applied over the whole brain in mice shortly before injecting the anti-PD-L1 IgG 89Zr-DFO-C4 or its FcRn low-affinity mutant 89Zr-DFO-C4Fc-MUT in a syngeneic glioblastoma murine model (GL261-GFP). Brain uptake was measured from PET scans acquired up to 7 days post-injection. Kinetic modeling was performed to compare the brain kinetics of both C4 formats. Results: FUS efficiently enhanced the delivery of both C4 radioligands in the brain with high reproducibility. 89Zr-DFO-C4Fc-MUT mean concentrations in the brain reached a significant uptake of 3.75±0.41%ID/cc with FUS against 1.92±0.45%ID/cc without, at 1h post-injection. A substantial and similar entry of both C4 radioligands was observed at a rate of 0.163±0.071 mL/h/g of tissue during 10.4±4.6min. The impaired interaction with FcRn of 89Zr-DFO-C4Fc-MUT significantly decreased the efflux constant from the healthy brain tissue to plasma compared with non-mutated IgG. Abolishing FcRn interaction allows determining the target engagement related to the specific binding as soon as 12h post-injection. Conclusion: Abolishing Fc-FcRn interaction confers improved kinetic properties to 89Zr-DFO-C4Fc-MUT for immunoPET imaging. FUS-aided BBB/BTB disruption enables quantitative imaging of PD-L1 expression by glioblastoma tumors within the brain.

SUPPLEMENTAL METHODS Radiochemical purity.Assessment of radiochemical purity by HPLC after radiolabeling of the C4 ligands with 89 Zr.Ex vivo biodistribution.After the last PET acquisition, under deep anesthesia, blood was collected from mice by cardiac puncture in heparinized tubes.Plasma was collected after centrifugation (10 min, 4000 rpm, 4°C).Heart, liver, spleen, kidney, muscle, and bone were collected, weighted and their activity was measured with a Cobra gamma counter (Packard).Background noise and decay correction were applied to the activity measurements.Then injected dose and weight of the organ corrections were performed to obtain data in injected dose per volume of tissue (%ID/cc).

FUS device.
Scheme S1.Scheme of the device used to disrupt mice's blood-brain-barrier and of the transducer trajectory on mice skulls.

Blood kinetic equations.
A two-compartment model with intravenous bolus input and first-order elimination was used to fit the plasma time-activity curves of mice injected with the C4 radioligands.Equations of the model are: And, With Vc: volume of the central compartment; k10: elimination constant from the central comportment; k12: transfer constant from the central to the peripheral compartment; k21: transfer constant from peripheral to central compartment; Vp: volume of the peripheral compartment.m1TCM and m2TCM equations.A discontinuous entry function was introduced to fit the IgG brain kinetic in area where FUS were applied.m1TCM equations are: With CTissue the concentration of 89 Zr-DFO-IgG in the considered tissue, K1 (mL/h/g of tissue) a perfusion-dependent entry constant, k2 (h -1 ) a transfer constant, TFUS (h) the time of closure of the BBB and KFUS (mL/h/g of tissue) the perfusion-dependent transfer constant when the BBB is disrupted.vB (%) is blood volume fraction.

Fig. S2 .
Fig. S2.Reversibility of the blood-brain barrier opening by FUS assessed by [ 18 F]2-fluoro-2deoxy-sorbitol ([ 18 F]FDS) PET imaging.BBB disruption was induced by FUS on the left hemisphere, and [ 18 F]FDS was intravenously injected immediately after (6.4±0.7 MBq, n = 5) and 24h after (6.1±0.4MBq, n=5) in the same C57Bl/6 healthy mice.FUS and PET imaging protocols are described by Hugon et al. (A) Standardized uptake value (SUV) normalized brain PET images of [ 18 F]FDS uptake 20 minutes and 24 hours after hemispheric BBB disruption induced by FUS.(B) Total volume of distribution (VT) was estimated using the Logan graphical analysis.Statistical significance was determined using a t-test with **** p-value < 0.0001.Data are represented as mean ± SD.

Fig. S3 .
Fig. S3.Scatter plot and regression lines (solid) of IgG C4 observed and predicted concentrations in blood (A), and brain regions: contralateral hemisphere (B), PET CE (C), T1w MRI CE (D).The dotted lines represent the line of identity.

Fig. S4 .
Fig. S4.Scatter plot and regression lines (solid) of IgG C4 Fc-MUT observed and predicted concentrations in blood (A), and brain regions: contralateral hemisphere (B), PET CE (C), T1w MRI CE (D).The dotted lines represent the line of identity. 1

Fig. S5
Fig. S5 Elements to evaluate accuracy of the fit models according to the entry function applied in animal injected with IgG C4 Fc-MUT in the T1w MRI contrast enhanced brain region.For each individual and model (left to right): graphic of predicted (blue line) and observed (red circle) concentrations during the first hour post-injection; graphic of associated residuals (grey circle) according to the time post-injection.AIC: Akaike criterion; SBC: Schwarz Bayesian criterion.

Fig. S6
Fig. S6 Elements to evaluate accuracy of the fit models according to the entry function applied in animal injected with IgG C4.For each individual and model (left to right): graphic of predicted (blue line) and observed (red circle) concentrations during the first hour post-injection; graphic of associated residuals (grey circle) according to the time post-injection.AIC: Akaike criterion; SBC: Schwarz Bayesian criterion.

Fig. S9 .
Fig. S9.Immunofluorescence and hematoxylin/eosin staining of brain sections of C4 Fc-MUT injected mice.(A) Adjacent 10µm cryo-sections were stained with either a rat anti-mouse-PD-L1 IgG and an AF546-goat-anti-rat IgG (orange) or AF546-goat-anti-human IgG (red) to detect the injected antibody.The immunofluorescence signal is overlaid on DAPI images (blue).Hematoxylin/eosin staining of adjacent cryo-sections.(B) Hematoxylin/eosin staining of adjacent sections of the ones in Figure 2C of the main manuscript.Scale bar = 1.0 mm.